Glioblastoma, the most common and deadly primary brain tumor, disseminates widely throughout the brain by hijacking the cell migration pathways used by normal neural stem cells. Migrating glioblastoma persist in the surrounding brain (pre-malignant field) after tumor resection, ultimately recurring and killing the patient. Glioblastoma migration is the hallmark of this devastating disease, yet no one has isolated and analyzed the single cell transcriptome of migrating glioblastoma as compared to normal (mature and fetal) or peritumoral astrocytes. We propose that migrating glioblastoma and peritumoral astrocytes employ fetal astrocyte genes to promote glioblastoma migration, that these genes are consistent within and across patients, and that inhibition of these genes will halt glioblastoma migration. To deconstruct and target glioblastoma migrating within the human peritumoral astrocyte microniche and suggest personalized therapies for patients, we have developed three innovative methods that leverage primary human tissue. Aim 1: To determine the extent to which migrating glioblastoma are defined by human fetal astrocyte gene expression, distinct from normal and glioblastoma astrocytes. Single cell isolation, RNA-seq, and transcriptome analysis of matched human glioblastoma, peritumoral, and normal brain will be used to identify brain cellular subtypes and migrating glioblastoma within the peritumoral brain. Migrating glioblastoma genetic markers that overlap with fetal astrocyte genes and are consistent within and across samples will be further validated. Aim 2: To test the extent to which peritumoral astrocytes facilitate glioblastoma migration through fetal astrocyte gene expression. Matched human glioblastoma and peritumoral astrocytes, isolated from fresh surgical specimens through our novel immunopanning separation technique, will undergo RNA-seq and culture. We suspect transcriptional and functional similarities between peritumoral astrocytes and normal fetal astrocytes. Candidate pathways will be promoted or inhibited in transwell migration assays using primary human glioblastoma. Aim 3: To test whether glioblastoma migration can be inhibited through knockdown of either fetal astrocyte genes in migrating glioblastoma or peritumoral astrocytes. Candidate genes identified in migrating glioblastoma (Aim 1) and peritumoral astrocytes (Aim 2) hold therapeutic promise, and will first be validated using primary human glioblastoma in vitro and ex vivo. Targets showing promise in these validation studies of either migrating glioblastoma or peritumoral astrocytes will undergo human glioblastoma- in-mouse intracranial xenograft modeling. Control and primary specimens will be imaged with CLARITY to confirm the dynamic glioblastoma-peritumoral astrocyte interactions. This project has direct translational potential as targeting glioblastoma migration will confine glioblastoma to a local disease, improving response to surgical resection and radiation by decreasing malignant progression.